This section introduces aspects that may help facilitate a better understanding of the disclosure. Accordingly, these statements are to be read in this light and are not to be understood as admissions about what is or is not prior art.
Ubiquitination has long been demonstrated to regulate the fate of numerous cellular proteins and recently it has become apparent that many GTPases, along with their GAPs, GeFs and GDis, undergo ubiquitination leading to a variety of fates such as re-localization or degradation.
Briefly, ubiquitin conjugation plays numerous intracellular roles such as localization, protein interactions, signaling and degradation. Therefore, targeting this process may represent an alternative approach. Additionally, deubiquitinating enzymes (DUBs), which remove ubiquitin from substrate proteins, may also be possible targets for therapeutic intervention and, although not yet at the clinical stage, the development of DUB inhibitors is indeed underway. Directly targeting the proteasome in order to prevent protein degradation has shown some promise, although due to the broad specificity ‘off-targets’ effects are likely. However, manipulating GTPase function by targeting ubiquitination specifically may be a way around this and produce a source of new therapeutic targets.
Ubiquitination is a reversible multistep process catalyzed by a number of enzymes, that entails the addition of a 79 amino acid tag to a substrate and while frequently this labels for destruction, it also may direct trafficking or alter function. Briefly, ubiquitin is initially activated by an E1 ubiquitin-activating enzyme in an energy dependent process requiring ATP and is subsequently transferred to an E2 ubiquitin conjugating enzyme. Finally, the ubiquitin is transferred to a lysine residue on the target protein with the assistance of a third enzyme termed the E3 ubiquitin ligase.
The removal of ubiquitin by DUBs alters the fate of the protein not only by rescuing it from degradation but also by altering its function, trafficking or activity. Although almost 100 human DUBs have been identified, this is a limited number compared with both the number of E3 ligases (>500) and ubiquitinated substrates suggesting that many of these enzymes must have multiple substrates. Although Ras and other GTPases are mutated in many common cancers, GTPases are by no means the only proteins modified by ubiquitin that are deregulated in cancer or other pathologies. But development of novel drugs to control their ubiquitin-modified activity has the potential to prove clinically beneficial.
A previous example, Bortezomib/Velcade that inhibits proteasome activity, has been approved for the treatment of multiple myeloma and is in clinical trials for other diseases such as non-small cell lung cancer, androgen-independent prostate carcinoma and non-Hodgkin's lymphoma and may also be useful in Ras or Rho mutant cancers. One concern with this broad spectrum approach is the potential side effects, and so more specific targeting of upstream proteins may prove a better option. E3 ligases are one possibility. Indeed, inhibitors for the HECT E3 ligase HDM2, key in p53 stability, have been identified. Additionally, small molecule inhibitors targeting DUB s are also under investigation. However, directly targeting the GTPases and their regulators, may prevent the side effects that have been observed with less specific drugs.
Therefore, there is a need to identify more specific upstream regulators for substrate ubiquitination.